CN101861331B

CN101861331B - Hypoallergenic variants of the major allergen from betula verrucosa pollen

Info

Publication number: CN101861331B
Application number: CN200880116680.6A
Authority: CN
Inventors: G·米斯特雷洛; S·扎诺塔; D·龙卡罗洛; P·法拉贾尼
Original assignee: Lofarma SpA
Current assignee: Lofarma SpA
Priority date: 2007-09-19
Filing date: 2008-09-16
Publication date: 2014-07-16
Anticipated expiration: 2028-09-16
Also published as: US8591907B2; CN101861331A; EA019696B1; EP2201031A1; AU2008300864B2; ITMI20071819A1; WO2009036949A1; MX2010002997A; US20100310590A1; CA2700047A1; CA2700047C; ES2460573T3; AU2008300864A1; EA201000360A1; EP2201031B1; DK2201031T3

Abstract

The invention provides hypoallergenic variants of Bet v 2 major allergen from Betula verrucosa plant pollen and the use thereof for the preventive or therapeutic treatment of allergic diseases.

Description

The main allergenic low allergenicity variant of betula pendula pollen

The invention provides the low allergenicity sequence variants of Bet v 2 protein, its nucleic acid molecule of encoding, the pharmaceutical composition that comprises it and they purposes in the allergic disease that prevention and treatment are caused by the plant pollen from betula pendula (Betulaverrucosa) species.

Background technology

Transformation reactions is caused extremely by function of immune system, and described immunity system is by producing the harmless proteins react containing in IgE-antibody-like and pollen, mite, epithelium and some food.

Nearest data show in western countries to surpass 10% population and suffer from this disease, and its symptom can worsen in time, for example cause asthma or to other allergen sensitization, cause thus and select suitable methods for the treatment of more difficult.

Different from pharmacological treatments, the immunotherapy that specifically desensitizes (SIT) is unique cause of disease therapy of allergic disease, can successfully change the characteristic immune parameter of this class disease.

Desensitization immunotherapy comprise use increase dosage available from the standard extract (vaccine) (1) that causes the material of disease.Like this, patient progressively reduces a para-immunity tolerance of this material, and allergic symptoms disappears subsequently.

But, in fact cause that the risk (2) of serious side effects limits the application of specific desensitization immunotherapy in treatment allergic disease, although the vaccine that uses slowly-releasing vaccine or use non-injecting pathway to use significantly reduces this risk.

Most of attention has concentrated on effective, the safe vaccine of exploitation in recent years, especially comprises the vaccine (3) of the recombinant protein (can advantageously affect the nature process of disease and do not cause the low allergenicity variant of undesirable side effect) of mutagenic treatment.

If IgG antibody is specific for sensitization allergen, one of favorable factor of SIT is induction.This class (protection) antibody can suppress antigen-IgE combination, particularly suppresses IgE and is combined with Bet v 2 antigens, changes the three-dimensional conformation (4,5) of this molecule.Comprise the exploitation with the vaccine that does not change immunogenic low allergenicity recombinant protein and will improve the treatment of allergic disease.

The pollen that is called the plant (birch, alder, hazel, Oak Tree, hornbeam) of Fagales (Fagales) on taxonomy is one of Temperate Region in China main reason of causing rhinallergosis and asthma.Two kinds of main allergens of birch pollen, Bet v 1 (being kept at the cDNA of GenBank with accession number X15877) and Bet v 2 (accession number M65179), be that molecular weight is respectively 17 and the protein (6,7) of 14kD.Bet v2 belongs to arrestin family, its relate to regulate eukaryotic cell cytoskeleton all in cytoplasmic protein matter.They and at least two kinds of cells (greatly) interaction of molecules, this at least two kinds of cells (greatly) molecule is phosphatidylinositols-4,5-bisphosphate, thereby stop C-γ Phospholipid hydrolase (phospholypase) to the hydrolysis of this lipid acid (8), and Actin muscle, regulate and control its polymerization (9).They participate in regulating microfilament precursor the high expression level hint of arrestin in pollen ripe and that sprout, and this microfilament precursor participates in germination process (10).Arrestin is considered to from the allergen in multiple arbor and herbal pollen, with the allergen in various fruits and vegetables, therefore cast aside the fact that arrestin is only found in 20% pair of pollen allergic (allergy) patient, they are defined as " general-allergen (pan-allergens) " (11,12).

High sequence homology (higher than 60%) in the plant arrestin in the multiple source of major part causes crossed sensitization, not only between from corresponding plants (13) and the pollen of uncorrelated plant (14), also at pollen and vegetable food product (aliment, 15) between, or between pollen and rubber (latex) (16).Homology between plant arrestin and Mammals arrestin is lower, although proved that they can and demonstrate interchangeability (17,18,19) in conjunction with the Actin muscle from different plant species.Explanation is that all arrestin have similar three-dimensional structure, as used (20,21,22) as shown in X-radiocrystallography.

Many immune equivalents that studies confirm that arrestin.In fact, having shown can be in conjunction with the arrestin of different sources from the patient's of the pollen sensitization to definite IgE, in conjunction with the IgE of arrestin, can mutually suppress (16).

High cross reactivity between different pollen makes independent arrestin can be used for allergic effect reaction diagnosis, and restructuring Bet v 2 is typically used as the allergen (23,24) of selecting arrestin specific IgE to measure.

There is the research of many measure arrestin IgE epi-position.

According to the contemporaneously following research (26) carried out of identical group, Vrtala (1996) is at Phe44 and Gln47 mutagenesis Bet v 2, they are become to Tyr44, Glu47 and Asn47, in described research, identified the linear epitope of monoclonal antibody 4A6 identification.Use the epitope mapping of synthetic dodecapeptide (crossing over the whole aminoacid sequence of Bet v2) to this antibody recognition.More effectively the polypeptide in conjunction with this antibody contains the region between amino acid 38-49 and 40-51.In the combination of IgG-peptide, the importance of Gln47 residue can not be identified the arrestin from tobacco (Nicotiana tabacum) and thimothy grass (Phleum pratense) by following evidence prove: 4A6, and its sequence shows that L-glutamic acid is positioned at the Gln47 of Bet v 2.Different from Gln47 → Glu47 sudden change, the change from Phe44 to Tyr44 or the change from GIn47 to Asn47 do not affect the combination of antibody.As shown in the immune marking and ELISA test (25), the mutagenesis identical to restructuring Bet v 2 application (Gln47 is to Glu, or Asn and Phe44 are to Tyr44) can not reduce the combination between arrestin and IgE.

In disclosed research (22) subsequently in 1997, by the random fragment of clone birch arrestin cDNA, identified main IgE epi-position, the expression library that this cDNA uses by oneself the allergic patients serum of arrestin is analyzed.Prove more responding property of three regions, corresponding to the region that is positioned at N-terminal α spiral (aa 1-30).The region of the α spiral of carboxyl terminal (aa 106-132), and the region of the fragment that comprises residue 30-50.

In research (23) subsequently, the comparison between the theoretical construct model of the arrestin to the research of IgE epi-position based on from different plants and birch or rubber arrestin crystal.Predicted 11 possible conformational epitopes (comprising adjacent amino acid region), wherein at least 20% is exposed to surface.Aminoacid sequence and conformation model relatively obtain two class epi-positions: species specificity epi-position (being characterized by high variability) and high conservative epi-position, the latter more may relate to the cross reaction between different plant arrestin.The result of aminoacid sequence comparison reflection Fedorov research (22), proves at arrestin N-end and has two possible linear epitopes, have three possible linear epitopes, and all the other two is held between residue 30-80 at arrestin C-.In the plant arrestin that all these regions are evaluated and tested in this research, are all high conservatives.The analysis of the 3D model of the prediction of possible conformational epitope based on rubber arrestin Hev b8.This analytical proof be selected from table centrical 12 outstanding residues.Although all possible epi-position is all imaginations, they comprise linear order and conservative or variable residue.The test of the specific IgE-binding ability of the epi-position that report confirmation is not studied in this research.

More recent publication relates to the evaluation (27) of muskmelon (Cucumis melo) arrestin IgE epi-position.The IgE of peptide of whole aminoacid sequence that crosses over this protein by mensuration is reactive, two linear epitopes have been identified, wherein E1 is identified strongly to the allergic patients serum of muskmelon, comprises residue 66-75 and 81-93, and E2 is comprised of amino acid 95-99 and 122-131.Two other epi-position is replied sign by weak IgE, i.e. E3 (residue 2-10) and E4 (35-45).The overlapping expression of the epi-position E1 of corresponding muskmelon arrestin 3D model and the peptide of E2 has two region: E1 and the E2 of the electrostatic property of good definition, and it is relevant with electronegativity protein domain with positive polarity respectively.

Data representation IgE arrestin epi-position from document is positioned at molecular moiety wherein, but does not point out to relate to the amino acid of IgE combination.

Summary of the invention

Having now found that by the one or more amino-acid residues that replace or lack in Bet v 2 allergen sequences makes this allergen reduce the reactivity of IgE antibody.

First aspect, the invention provides low allergenicity protein, and it is the allergenic sequence variants of Bet v 2, and it is characterized in that:

1) compare with wild-type Bet v 2 allergens (SEQ ID NO:1), the reactivity of IgE is reduced;

2) aminoacid sequence is:

A) identical with SEQ ID NO:1 at least 90%, preferably at least 93% is identical, further preferentially at least 97% identical;

B) show that with the sequence alignment of SEQ ID NO:1 SEQ ID NO:1 is corresponding to (matching) Ser ₃₉, Lys ₄₅, Lys ₈₈or Lys ₈₉ser or Lys residue place there is at least one replacement or disappearance.

In preferred embodiments, the allergenic variant of Bet v 2 of the present invention shown in position there is a plurality of replacements or the disappearance in 1-3, wherein produce single, double or three replace and/or disappearance variant.Although can there is replacement and the disappearance of different aminoacids residue in Bet v 2 molecules simultaneously, preferably indicating site by the monosubstituted acquisition variant of one or more residues, particularly this type of residue is by the variant of neutrality, polarity or acidic amino acid displacement, described neutrality, polarity or acidic amino acid are selected from Ala, Thr, Gly, Pro, Leu, Ile, Ser, Phe, Glu, Asp, are more preferably selected from Ala, Thr, Ser, Gly, Glu, Asp.

The example of variant of the present invention is shown in SEQ ID NO:2 (1 residue replaces), SEQ ID NO:3 (1 residue replaces), SEQ ID NO:4 (2 residues replace), SEQ ID NO:5 (3 residues replace) and SEQ ID NO:6 (3 residues replace).

With respect to wild type counterparts (counterpart), Bet v of the present invention 2 allergens replace and/or disappearance variant shows the reactivity with betula pendula pollen-allergy patients serum's IgE, it reduces at least 10%, preferably at least 50%, more preferably at least 90%, wherein IgE reactivity detects by for example ELISA method for measuring.

IgE reactivity from the protein s EQ ID NO:2-6 in allergy patients serum storehouse is measured and is detected (Fig. 1) by ELISA.With respect to wild-type Bet v 2 allergens (SEQ ID NO:1), when (SEQID NO:2), (SEQ ID NO:3), (SEQ ID NO:4) and (SEQ ID NO:5 and 6) protein are when hatching from birch pollen allergy patient's Serum Bank, observe reactive average 92% (SEQ IDNO:2), 13% (SEQ ID NO:3), 97% (SEQ ID NO:4) and 93% (the SEQ ID NO:5 and 6) of reducing of IgE.

These results confirm by REAST inhibition test, and this test can be evaluated and tested the reactivity from the homology epi-position of different proteins.When there is 1.45ng inhibition, when serum pretreated with identical protein, wild-type Bet v 2 (SEQ ID NO:1) suppresses with the combination 82.6% from the IgE in allergy patients serum storehouse, when serum is used variant SEQ ID NO:2 and SEQ ID NO:3 preincubation, it is respectively 40.4% and 71% inhibition, and when serum uses respectively two replacement variants (SEQ IDNO:4) and three of same amount to replace variant (SEQ ID NO:5 and SEQ ID NO:6) preincubation, the inhibition of observing is only 13.4%, 4% and 8.8% (Fig. 2).

These results clearly show that the amino acid of the 39th, 45,88,89 of SEQ ID NO:1 participates in IgE to the allergenic identification of Bet v 2.

In addition, Balb/c mouse immune inoculation test proof wild-type Bet v 2 allergens and low allergenicity protein s EQ ID NO:5 (being selected from as exemplary sudden change allergen) all can induce IgG-specific immune response (Fig. 3).Anti-SEQ ID NO:5 antibody can be identified wild-type-counterpart SEQ IDNO:1 (Fig. 4), proves that the replacement of the 45th, 88,89 Lys-residues does not cause the great change of molecular immune originality, and changes its IgG epi-position.On the contrary, the antibody existing in the mice serum of uncorrelated antigen immune can not be identified wild-type Bet v 2 and SEQ ID NO:5.

On the other hand, the invention provides the corresponding immune-active peptides of Bet v 2 fragment, described immune-active peptides comprises at least one above-mentioned replacement and/or disappearance.Described peptide preferably contains 15-35 amino-acid residue, more preferably 15-20 amino-acid residue.Term used herein " immune-active peptides " means to cause the peptide of the immunne response that does not rely on IgE.

Use method known to those skilled in the art and technology, by mutagenesis wild-type Bet v 2cDNA sequence (SEQ ID NO:7), can easily prepare replacement of the present invention and/or disappearance variant.

Single, double and the three cDNA sequences that replace variant shown in coding SEQ ID NO:2-6 are reported in SEQ ID NO:8-12.

On the other hand, the invention provides the nucleic acid molecule of coding low allergenicity Bet v disclosed herein 2 variants, derived from its peptide, the expression vector that comprises this molecule, in wherein said molecule and eukaryotic cell or prokaryotic cell prokaryocyte, control the functional connection of genetic elements (for example transcripting promoter, enhanser, signal and leader sequence, or other sequences that relate in transcriptional control) of its expression.The example of carrier comprises plasmid, virus and phage, yet in genetic engineering, normally used any other carrier also can be used.

The present invention also comprises protokaryon or the eukaryotic host cell with carrier conversion of the present invention or transfection.Prokaryotic cell prokaryocyte is intestinal bacteria (Escherichia coli) or Bacillus subtilus (Bacillus subtilis) for example, or eukaryotic cell for example yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) be typically used as clone or cDNA expression vector.

In addition, low allergenicity variant of the present invention can be used as fusion rotein preparation.

Because the IgE that they reduce is reactive, Bet v 2 variants of the present invention can be advantageously used in the preparation of pharmaceutical composition (for example tablet), and described pharmaceutical composition is for preventing or treat the allergy individuality of betula pendula pollen.

Therefore another aspect of the present invention is pharmaceutical composition, low allergenicity Bet v provided herein 2 variants that it comprises significant quantity, other allergens of the optional and betula pendula of this variant, and/or pharmaceutical acceptable carrier and excipient composition.In preferred embodiments, pharmaceutical composition is used for preventing or treating with vaccine form the allergic disease that comprises bronchial asthma, rhinallergosis, allergic dermatitis and allergic conjunctivitis.The theory of immunization and operation are (28,29) well known by persons skilled in the art.

The following example is further illustrated the present invention.Unless pointed out separately, in embodiment, method therefor is described in Sambrook, Fritsch ET Maniatis " Molecular cloning.A laboratorymanual " second edition, and 1-2-3 volume, CSH Lab publishes, and 1989.

Accompanying drawing summary

Fig. 1: the reactivity of elisa assay IgE and Bet v 2 allergens and Bet v 2 low allergenicity variants;

The inhibition that Fig. 2: IgE is combined with Betv 2 allergens;

Fig. 3: the replying of sudden change IgE and each immunogenic protein;

Fig. 4: the IgG in the mouse of SEQ ID NO:5 immunity replys.

The site-specific mutagenesis of embodiment 1-Bet v 2 allergen code cDNAs

The site-specific mutagenesis of Bet v 2 allergen code cDNAs (SEQ ID NO:7), by the cDNA clone in prokaryotic vector (pBluescript, GenBank accession number X52327), is then that pcr amplification carries out.The oligonucleotide (table) that is used as primer in PCR reaction has suitable base and replaces.For each mutagenesis, use the complementary oligonucleotide (30) that is attached to DNA chain respective regions.After amplification, the unaltered original template of enzymatic digestion degradation selectivity of restriction enzyme DpnI catalysis.Then with the molecule of mutagenesis, transform Bacillus coli cells.According to mulberry lattice (method), the clone available from single bacteria bacterium colony is checked order, to determine base modification correct in cDNA and not have non-specific sudden change.

Table. in site-specific mutagenesis, be used as the oligonucleotide sequence of primer.

The base of sudden change is black matrix.

Oligonucleotide	Sequence
		Bet v2 S39	ggg ccc aga gcg ctt cct tcc cac ag
Bet v2 K45	cct tcc cac agt tta cgc ctc agg aaa tc
		Bet v2 K88-89	gtc atc cgt gga ggg gag gga tct gga g

Embodiment 2-prepares Bet v 2 albumen and variant thereof

According to standard scheme (31,32), in intestinal bacteria, clone and express (SEQ ID NO:8-12) Bet v 2cDNA of wild-type (SEQ ID NO.7) and mutagenesis.Cell centrifugation is collected, and is resuspended in PBS1X (6.46mM NaH ₂pO ₄, 1.47mM KH ₂pO ₄, 136.89mM NaCl) in and ultrasonic degradation.Centrifugation recombinant protein.The precipitation that contains insoluble protein aggregate is resuspended in PBS 1X, 6M urea (sex change damping fluid) and at 4 ℃, stirs 60 minutes.Recombinant protein centrifugation from insoluble residue of dissolving, with PBS 1X dialysis, the filter that filters 1 μ M is also used agarose column (Sigma, Milan, the Italy) affinitive layer purification of poly--proline(Pro) derivatize by use.With after PBS 1X, the washing of 2M urea, with PBS 1X, 8M urea wash-out recombinant protein, and 4 ℃ of dialysis carry out refolding for 16 hours in PBS 1X solution.

The sign of embodiment 3-allergy experimenter serum

Serum is collected from having has the clinical patient history of seasonal allergy to betula pendula pollen, and has the Bet v original RAST 3+ of 2 allergic effect and the atopic individuality of 4+, then concentrates these serum and uses with this form.Serum Bank from anallergic patient is used as negative control.

The reactive elisa assay of embodiment 4-Bet v 2 variants to IgE in Serum Bank

In 50mM carbonate/bicarbonate damping fluid (pH 9.6) the wild-type allergen of same amount and mutation variants (1 μ g) at 4 ℃ incubation 16 hours to be adsorbed onto on the hole of polystyrene for elisa assay (polystirene) plate.This is the washing soln (phosphate buffered saline buffer that 60mM contains 0.05%Tween-20 for hole, pH 6.5) washing, and seal with diluting soln (25% lowlenthal serum in 150mM phosphate buffered saline buffer, 1mM EDTA, 0.05%Tween 20,0.01% thiomersal(ate), pH 7.4).The aliquot sample of 70 μ l human serum RAST 3+ and 4+ storehouse (in dilution buffer liquid) is added to each sample, and in 25 ℃ of incubations 2 hours.Wash after three times, add peroxidase conjugated anti-human-IgE serum (in dilution buffer liquid 1: 1500), then 25 ℃ of incubations are 1.5 hours.After washing three times, add 100 μ l TMB reagent (BioFX Laboratories, Owings Mills, MD) and within 15 minutes, carry out colorimetric reaction at 25 ℃ of incubations.Add 100 μ l 1N HCl termination reactions and use microplate reader (microplate reader spectrophotometer) at 450nm place reading.

Embodiment 5-REAST suppresses to measure.The combination of the IgE comprising in Bet v 2 variants inhibition biotinylation Bet v 2 and Serum Bank

Be diluted at 1: 3 in dilution buffer liquid (25% lowlenthal serum in 150mM phosphate buffered saline buffer, 1mM EDTA, 0.05%Tween 20,0.01% thiomersal(ate), pH 7.4) to the preincubation 1.5 hours 25 ℃ of the serial dilution thing (starting from 67ng/ml) of the aliquot sample of the human serum storehouse RAST 4+ of Bet v 2 and 3+ (50 μ l) and wild-type allergen and mutant thereof.Then mixture is added in the dull and stereotyped aperture of ELISA polystyrene (polystirene) and at 25 ℃ of incubations 1.5 hours, the anti-IgE of this aperture absorption people.With phosphoric acid buffer, the 0.05%Tween-20 of 0.06M, after pH6.5 washing three times, add 0.1ml biotinylation Bet v 2 antigens (85.3ng/ml) in dilution buffer liquid and 25 ℃ of incubations 1 hour.Wash after three times, add peroxidase-Streptavidin (0.1 μ g/ml) 30 minutes at 25 ℃.With 100 μ l 1N HCl, carry out colorimetric reaction and use spectrophotometer at 450nm place reading.

Suppressing percentage ratio is calculated as follows: 100x[(A-B)/A], the absorbancy that when wherein A is unrestraint thing, 450nm place is measured, and B is the absorbancy while there is inhibition.

The scheme of embodiment 6-immunization Balb/c mouse

The emulsion subcutaneous inoculation of 200 μ l for two groups of 5 female Balb/c mouse (Charles River), this emulsion contains the 20 μ g antigens (SEQ IDNO:1, SEQ ID NO:5) in 100 μ l complete Freund's adjuvants and 100 μ l salt solution.Other three booster immunizations carry out with the interval of 1 week, and the full adjuvant that toos many or too much for use is replaced Freund's complete adjuvant.In contrast, 5 mouse are used incoherent antigen.Latter 7 days of last immunity, obtains blood sample from jugular vein, the replying with check antibody and every kind of immunogenic substance for ELISA.For the mouse of SEQ ID NO:5 immunity, also analyzed the ability of identification wild-type protein.

The IgG-specificity that embodiment 7-ELISA analyzes in immune mouse is replied

In 50mM carbonate/bicarbonate damping fluid (pH 9.6), the wild-type Bet v 2 of same amount and variant SEQ ID NO:5 (0.25 μ g) incubation at 4 ℃ is used on the hole of polystyrene board to be adsorbed onto elisa assay for 16 hours.This is the washing soln (phosphate buffered saline buffer that 60mM contains 0.05%Tween-20 for hole, pH 6.5) washing, and seal with diluting soln (25% horse serum in 150mM phosphate buffered saline buffer, 1mM EDTA, 0.05%Tween 20,0.01% thiomersal(ate), pH 7.4).The aliquot sample of the serial dilution thing (in dilution buffer liquid) of every mice serum of 100 μ l is placed in every hole, and in 25 ℃ of incubations 2 hours.

Wash after three times, the anti-mouse IgG serum of 1: 2000 dilution peroxidase conjugated in dilution buffer liquid, and join in aperture, follow 25 ℃ of incubations 1.5 hours.After washing three times, add 100 μ lTMB reagent (BioFX Laboratories, Owings Mills, MD) and within 15 minutes, carry out colorimetric reaction at 25 ℃ of incubations.By 100 μ l 1N HCl termination reactions and use spectrophotometer at 450nm place reading.Fig. 3 and 4 shows the average response obtaining by analyzing the serum of 5 mouse of every group.

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32)Asturias JA，lbarrola I，Eseverri JL，Arilla MC，Gonzales-Rioja R，Martinez A.(2004).“PCR-based cloning and immunological characterizationof Parietaria judaica pollen profilin”.J Investig Allergol Clin Immunol，14：43-48.

Sequence table

<110> Lofarma S. P. A. (LOFARMA S.P.A.)

The main allergenic low allergenicity variant of <120> betula pendula pollen

<130>8025MEUR

<160>12

<170>PatentIn version 3 .3

<210>1

<211>133

<212>PRT

<213> is unknown

<220>

<223> is from the wild-type Bet v 2 of betula pendula (Betula verrucosa)

<400>1

Met Ser Trp Gln Thr Tyr Val Asp Glu His Leu Met Cys Asp Ile Asp

1 5 10 15

Gly Gln Ala Ser Asn Ser Leu Ala Ser Ala Ile Val Gly His Asp Gly

20 25 30

Ser Val Trp Ala Gln Ser Ser Ser Phe Pro Gln Phe Lys Pro Gln Glu

35 40 45

Ile Thr Gly Ile Met Lys Asp Phe Glu Glu Pro Gly His Leu Ala Pro

50 55 60

Thr Gly Leu His Leu Gly Gly Ile Lys Tyr Met Val Ile Gln Gly Glu

65 70 75 80

Ala Gly Ala Val Ile Arg Gly Lys Lys Gly Ser Gly Gly Ile Thr Ile

85 90 95

Lys Lys Thr Gly Gln Ala Leu Val Phe Gly Ile Tyr Glu Glu Pro Val

100 105 110

Thr Pro Gly Gln Cys Asn Met Val Val Glu Arg Leu Gly Asp Tyr Leu

115 120 125

Ile Asp Gln Gly Leu

130

<210>2

<211>133

<212>PRT

<213> is unknown

<220>

<223>Bet v 2 mutant

<400>2

Met Ser Trp Gln Thr Tyr Val Asp Glu His Leu Met Cys Asp Ile Asp

1 5 10 15

Gly Gln Ala Ser Asn Ser Leu Ala Ser Ala Ile Val Gly His Asp Gly

20 25 30

Ser Val Trp Ala Gln Ser Ala Ser Phe Pro Gln Phe Lys Pro Gln Glu

35 40 45

Ile Thr Gly Ile Met Lys Asp Phe Glu Glu Pro Gly His Leu Ala Pro

50 55 60

Thr Gly Leu His Leu Gly Gly Ile Lys Tyr Met Val Ile Gln Gly Glu

65 70 75 80

Ala Gly Ala Val Ile Arg Gly Lys Lys Gly Ser Gly Gly Ile Thr Ile

85 90 95

Lys Lys Thr Gly Gln Ala Leu Val Phe Gly Ile Tyr Glu Glu Pro Val

100 105 110

Thr Pro Gly Gln Cys Asn Met Val Val Glu Arg Leu Gly Asp Tyr Leu

115 120 125

Ile Asp Gln Gly Leu

130

<210>3

<211>133

<212>PRT

<213> is unknown

<220>

<223>Bet v 2 mutant

<400>3

Met Ser Trp Gln Thr Tyr Val Asp Glu His Leu Met Cys Asp Ile Asp

1 5 10 15

Gly Gln Ala Ser Asn Ser Leu Ala Ser Ala Ile Val Gly His Asp Gly

20 25 30

Ser Val Trp Ala Gln Ser Ser Ser Phe Pro Gln Phe Thr Pro Gln Glu

35 40 45

Ile Thr Gly Ile Met Lys Asp Phe Glu Glu Pro Gly His Leu Ala Pro

50 55 60

Thr Gly Leu His Leu Gly Gly Ile Lys Tyr Met Val Ile Gln Gly Glu

65 70 75 80

Ala Gly Ala Val Ile Arg Gly Lys Lys Gly Ser Gly Gly Ile Thr Ile

85 90 95

Lys Lys Thr Gly Gln Ala Leu Val Phe Gly Ile Tyr Glu Glu Pro Val

100 105 110

Thr Pro Gly Gln Cys Asn Met Val Val Glu Arg Leu Gly Asp Tyr Leu

115 120 125

Ile Asp Gln Gly Leu

130

<210>4

<211>133

<212>PRT

<213> is unknown

<220>

<223>Bet v 2 mutant

<400>4

Met Ser Trp Gln Thr Tyr Val Asp Glu His Leu Met Cys Asp Ile Asp

1 5 10 15

Gly Gln Ala Ser Asn Ser Leu Ala Ser Ala Ile Val Gly His Asp Gly

20 25 30

Ser Val Trp Ala Gln Ser Ser Ser Phe Pro Gln Phe Lys Pro Gln Glu

35 40 45

Ile Thr Gly Ile Met Lys Asp Phe Glu Glu Pro Gly His Leu Ala Pro

50 55 60

Thr Gly Leu His Leu Gly Gly Ile Lys Tyr Met Val Ile Gln Gly Glu

65 70 75 80

Ala Gly Ala Val Ile Arg Gly Gly Glu Gly Ser Gly Gly Ile Thr Ile

85 90 95

Lys Lys Thr Gly Gln Ala Leu Val Phe Gly Ile Tyr Glu Glu Pro Val

100 105 110

Thr Pro Gly Gln Cys Asn Met Val Val Glu Arg Leu Gly Asp Tyr Leu

115 120 125

Ile Asp Gln Gly Leu

130

<210>5

<211>133

<212>PRT

<213> is unknown

<220>

<223>Bet v 2 mutant

<400>5

Met Ser Trp Gln Thr Tyr Val Asp Glu His Leu Met Cys Asp Ile Asp

1 5 10 15

Gly Gln Ala Ser Asn Ser Leu Ala Ser Ala Ile Val Gly His Asp Gly

20 25 30

Ser Val Trp Ala Gln Ser Lys Ser Phe Pro Gln Phe Thr Pro Gln Glu

35 40 45

Ile Thr Gly Ile Met Lys Asp Phe Glu Glu Pro Gly His Leu Ala Pro

50 55 60

Thr Gly Leu His Leu Gly Gly Ile Lys Tyr Met Val Ile Gln Gly Glu

65 70 75 80

Ala Gly Ala Val Ile Arg Gly Gly Glu Gly Ser Gly Gly Ile Thr Ile

85 90 95

Lys Lys Thr Gly Gln Ala Leu Val Phe Gly Ile Tyr Glu Glu Pro Val

100 105 110

Thr Pro Gly Gln Cys Asn Met Val Val Glu Arg Leu Gly Asp Tyr Leu

115 120 125

Ile Asp Gln Gly Leu

130

<210>6

<211>133

<212>PRT

<213> is unknown

<220>

<223>Bet v 2 mutant

<400>6

Met Ser Trp Gln Thr Tyr Val Asp Glu His Leu Met Cys Asp Ile Asp

1 5 10 15

Gly Gln Ala Ser Asn Ser Leu Ala Ser Ala Ile Val Gly His Asp Gly

20 25 30

Ser Val Trp Ala Gln Ser Ala Ser Phe Pro Gln Phe Lys Pro Gln Glu

35 40 45

Ile Thr Gly Ile Met Lys Asp Phe Glu Glu Pro Gly His Leu Ala Pro

50 55 60

Thr Gly Leu His Leu Gly Gly Ile Lys Tyr Met Val Ile Gln Gly Glu

65 70 75 80

Ala Gly Ala Val Ile Arg Gly Gly Glu Gly Ser Gly Gly Ile Thr Ile

85 90 95

Lys Lys Thr Gly Gln Ala Leu Val Phe Gly Ile Tyr Glu Glu Pro Val

100 105 110

Thr Pro Gly Gln Cys Asn Met Val Val Glu Arg Leu Gly Asp Tyr Leu

115 120 125

Ile Asp Gln Gly Leu

130

<210>7

<211>402

<212>DNA

<213> is unknown

<220>

<223> is from the encoding sequence of the wild-type Bet v 2 of betula pendula

<400>7

atgtcgtggc aaacgtacgt ggatgaacat ttgatgtgcg atatcgacgg gcaagccagc 60

aactcgctgg catctgcgat cgtcggtcac gatggctctg tgtgggccca gagctcttcc 120

ttcccacagt ttaagcctca ggaaatcact ggtatcatga aggactttga ggagccgggt 180

catcttgctc cgacgggctt acaccttggg ggcataaaat acatggtcat ccagggagag 240

gctggtgctg tcatccgtgg aaagaaggga tctggaggta ttactataaa gaagactggt 300

caagctctcg tttttggcat ctatgaagag cctgtgacac caggacagtg caacatggtt 360

gttgagaggt tgggggatta ccttattgac cagggcctgt ag 402

<210>8

<211>402

<212>DNA

<213> is unknown

<220>

The encoding sequence of <223>Bet v 2 mutant

<400>8

atgtcgtggc aaacgtacgt ggatgaacat ttgatgtgcg atatcgacgg gcaagccagc 60

aactcgctgg catctgcgat cgtcggtcac gatggctctg tgtgggccca gagcgcttcc 120

ttcccacagt ttaagcctca ggaaatcact ggtatcatga aggactttga ggagccgggt 180

catcttgctc cgacgggctt acaccttggg ggcataaaat acatggtcat ccagggagag 240

gctggtgctg tcatccgtgg aaagaaggga tctggaggta ttactataaa gaagactggt 300

caagctctcg tttttggcat ctatgaagag cctgtgacac caggacagtg caacatggtt 360

gttgagaggt tgggggatta ccttattgac cagggcctgt ag 402

<210>9

<211>402

<212>DNA

<213> is unknown

<220>

The encoding sequence of <223>Bet v 2 mutant

<400>9

atgtcgtggc aaacgtacgt ggatgaacat ttgatgtgcg atatcgacgg gcaagccagc 60

aactcgctgg catctgcgat cgtcggtcac gatggctctg tgtgggccca gagctcttcc 120

ttcccacagt ttacgcctca ggaaatcact ggtatcatga aggactttga ggagccgggt 180

catcttgctc cgacgggctt acaccttggg ggcataaaat acatggtcat ccagggagag 240

gctggtgctg tcatccgtgg aaagaaggga tctggaggta ttactataaa gaagactggt 300

caagctctcg tttttggcat ctatgaagag cctgtgacac caggacagtg caacatggtt 360

gttgagaggt tgggggatta ccttattgac cagggcctgt ag 402

<210>10

<211>402

<212>DNA

<213> is unknown

<220>

The encoding sequence of <223>Bet v 2 mutant

<400>10

atgtcgtggc aaacgtacgt ggatgaacat ttgatgtgcg atatcgacgg gcaagccagc 60

aactcgctgg catctgcgat cgtcggtcac gatggctctg tgtgggccca gagctcttcc 120

ttcccacagt ttaagcctca ggaaatcact ggtatcatga aggactttga ggagccgggt 180

catcttgctc cgacgggctt acaccttggg ggcataaaat acatggtcat ccagggagag 240

gctggtgctg tcatccgtgg aggggaggga tctggaggta ttactataaa gaagactggt 300

caagctctcg tttttggcat ctatgaagag cctgtgacac caggacagtg caacatggtt 360

gttgagaggt tgggggatta ccttattgac cagggcctgt ag 402

<210>11

<211>402

<212>DNA

<213> is unknown

<220>

The encoding sequence of <223>Bet v 2 mutant

<400>11

atgtcgtggc aaacgtacgt ggatgaacat ttgatgtgcg atatcgacgg gcaagccagc 60

aactcgctgg catctgcgat cgtcggtcac gatggctctg tgtgggccca gagctcttcc 120

ttcccacagt ttacgcctca ggaaatcact ggtatcatga aggactttga ggagccgggt 180

catcttgctc cgacgggctt acaccttggg ggcataaaat acatggtcat ccagggagag 240

gctggtgctg tcatccgtgg aggggaggga tctggaggta ttactataaa gaagactggt 300

caagctctcg tttttggcat ctatgaagag cctgtgacac caggacagtg caacatggtt 360

gttgagaggt tgggggatta ccttattgac cagggcctgt ag 402

<210>12

<211>402

<212>DNA

<213> is unknown

<220>

The encoding sequence of <223>Bet v 2 mutant

<400>12

atgtcgtggc aaacgtacgt ggatgaacat ttgatgtgcg atatcgacgg gcaagccagc 60

aactcgctgg catctgcgat cgtcggtcac gatggctctg tgtgggccca gagcgcttcc 120

ttcccacagt ttaagcctca ggaaatcact ggtatcatga aggactttga ggagccgggt 180

catcttgctc cgacgggctt acaccttggg ggcataaaat acatggtcat ccagggagag 240

gctggtgctg tcatccgtgg aggggaggga tctggaggta ttactataaa gaagactggt 300

caagctctcg tttttggcat ctatgaagag cctgtgacac caggacagtg caacatggtt 360

gttgagaggt tgggggatta ccttattgac cagggcctgt ag 402

Claims

1. low allergenicity protein, it is the main allergenic sequence variants of Bet v2, wherein said protein:

A) with by the wild-type Bet v2 allergen shown in SEQ ID NO:1 compare, the reactivity of IgE is reduced; With

B) by least one position Ser at SEQ ID NO:1 ₃₉, Lys ₄₅, Lys ₈₈or Lys ₈₉ser or the replacement of Lys residue from SEQ ID NO:1, obtain, wherein said Ser or Lys residue are substituted by the amino-acid residue that is selected from Ala, Thr, Ser, Gly, Glu and Asp.

2. according to the low allergenicity protein of claim 1, it is selected from SEQ ID NO:2-6.

3. nucleic acid molecule, its coding claim 1 or 2 protein.

4. according to the nucleic acid molecule of claim 3, it is selected from SEQ ID NO:8-12.

5. carrier, the nucleic acid molecule that it contains claim 3 or 4.

6. host cell, the carrier that it contains claim 5.

7. pharmaceutical composition, its claim that contains significant quantity 1 or 2 low allergenicity protein, and pharmaceutically acceptable carrier.

8. pharmaceutical composition, its claim that contains significant quantity 1 or 2 low allergenicity protein, and vehicle.

9. according to the composition of claim 7 or 8, it is vaccine form.

10. the purposes in the pharmaceutical composition of the preventative or therapeutic treatment of the allergic disease that claim 1 or 2 low allergenicity protein cause at the plant pollen for the preparation of by from betula pendula species.

11. according to the purposes of claim 10, and wherein said allergic disease is selected from bronchial asthma and allergic conjunctivitis.